Objective
This experiment is designed to investigate the IC 50 of compounds which had the positive effect on the reduction of the viability of leukemia cells.
Introduction
IC 50 stands for “half maximum inhibitory concentration” (2). IC 50 represents the concentration of the compounds that required for 50% of cell death. The IC 50 assay is used to determine the potency of the drugs which used as treatment of cancers (1). In the previous experiments, 20,000 cells/mL was the optimal number of leukemia cells, which was the concentration of cells given the absorbance values between 0.75 and 1.25 in MTT assay. Moreover, compounds were tested for their possible effect on the reduction of the proliferation of leukemia cells. Compound 3, 5, 8 and …show more content…
In the lab, 250 mL of each concentration of the compounds was made. The serial dilution was performed as shown in the Figure 1. Cells were counted by using the hemocytometer to estimate the concentration of cells in the original culture flask. Cells were counted 2x106 cells/mL. The optimal number of cells determined from previous lab was 20,000 cells/ mL. Therefore, 10 µL of cells, 10 µL of compounds, and 90 µL of media was added into each well. The plates were incubated for 48 hours for the cells to stabilize and interact with compound.
After that, MTT assay was used to determine the viability of the cells. 10 µL of MTT reagent was inserted into each well. The plate was incubated for 2-4 hours more. Then, 100 µL of detergent reagent was added. The plates again were incubated for 2-4 hours. Finally, the plate was read at 570 nm using a spectrophotometer.
The graphs were plotted of the percentage of cell death versus concentration of the compounds. The logarithm trend-line and equation were included in the graphs. The IC 50 values of the compounds were determined based on the …show more content…
The cells should be equally distributed in the hemocytometer, so the original concentration of cells is more accurately determined. Cells need to be equally suspended in the media while seeding the cells into the wells to prevent the concentration of cells not being accurate because cells might clump together or stay at the bottom of the Eppendorf tube. The adding of reagent detergent step needs to be performed quickly, and do not shake the plate because when the plate stays too long outside, the light will affect the result.
Citation
(1) Beck B, Chen YF, Dere W, et al. (2012) Assay Operations for SAR Support. Retrieved April 20, 2017, from https://www.ncbi.nlm.nih.gov/books/NBK91994/
(2) IC50. (2017) In Wikipedia, The Free Encyclopedia. Retrieved 14:48, April 20, 2017, from https://en.wikipedia.org/w/index.php?title=IC50&oldid=773146178
(3) MV-4-11 (ATCC® CRL-9591™). Retrieved March 08, 2017, from
This experiment is designed to investigate the IC 50 of compounds which had the positive effect on the reduction of the viability of leukemia cells.
Introduction
IC 50 stands for “half maximum inhibitory concentration” (2). IC 50 represents the concentration of the compounds that required for 50% of cell death. The IC 50 assay is used to determine the potency of the drugs which used as treatment of cancers (1). In the previous experiments, 20,000 cells/mL was the optimal number of leukemia cells, which was the concentration of cells given the absorbance values between 0.75 and 1.25 in MTT assay. Moreover, compounds were tested for their possible effect on the reduction of the proliferation of leukemia cells. Compound 3, 5, 8 and …show more content…
In the lab, 250 mL of each concentration of the compounds was made. The serial dilution was performed as shown in the Figure 1. Cells were counted by using the hemocytometer to estimate the concentration of cells in the original culture flask. Cells were counted 2x106 cells/mL. The optimal number of cells determined from previous lab was 20,000 cells/ mL. Therefore, 10 µL of cells, 10 µL of compounds, and 90 µL of media was added into each well. The plates were incubated for 48 hours for the cells to stabilize and interact with compound.
After that, MTT assay was used to determine the viability of the cells. 10 µL of MTT reagent was inserted into each well. The plate was incubated for 2-4 hours more. Then, 100 µL of detergent reagent was added. The plates again were incubated for 2-4 hours. Finally, the plate was read at 570 nm using a spectrophotometer.
The graphs were plotted of the percentage of cell death versus concentration of the compounds. The logarithm trend-line and equation were included in the graphs. The IC 50 values of the compounds were determined based on the …show more content…
The cells should be equally distributed in the hemocytometer, so the original concentration of cells is more accurately determined. Cells need to be equally suspended in the media while seeding the cells into the wells to prevent the concentration of cells not being accurate because cells might clump together or stay at the bottom of the Eppendorf tube. The adding of reagent detergent step needs to be performed quickly, and do not shake the plate because when the plate stays too long outside, the light will affect the result.
Citation
(1) Beck B, Chen YF, Dere W, et al. (2012) Assay Operations for SAR Support. Retrieved April 20, 2017, from https://www.ncbi.nlm.nih.gov/books/NBK91994/
(2) IC50. (2017) In Wikipedia, The Free Encyclopedia. Retrieved 14:48, April 20, 2017, from https://en.wikipedia.org/w/index.php?title=IC50&oldid=773146178
(3) MV-4-11 (ATCC® CRL-9591™). Retrieved March 08, 2017, from